Bu çalışma Ulusal Sağlık Enstitüleri Sağlık hibe AI62983 (AYK), AI22535 (GPB) tarafından finanse edildi.

1. P. murin Modeli aeruginosa GI Kolonizasyon ve Yaygınlaştırma <ol><li> Komensal florası tüketmek ve daha sonra daha önce 4 açıklandığı gibi mono-PA ile kolonize C3H/HeN fareler (6-8 hafta eski kadın, Harlan) oral antibiyotik ile tedavi edilir. </li></ol> 2. Murin Çekal Luminal İçeriği Hasat <ol><li> Sıvı azot banyo temizlenir paslanmaz çelik harcı bırakın. </li><li> Karbondioksit boğulma tarafından fareler Euthanize. </li><li> Strafor kurulu ve% 95 etanol ile duş Güvenli fare karkas. Sternum perine deri yoluyla orta hat longitudinal kesi olun. Deri ve periton karın boşluğunda maruz yansıtmaktadır. </li><li> Tüm çekum rezeke. Çekum paslanmaz çelik harç üzerinde forseps ile tutun. Çekum diseksiyon makas ile her iki ucunda Snip. </li><li> Çekum proksimal ucunu çekal flushate tamponu (10 mM TrisHCl, 1 mM EDTA ve 200 mM NaCl) 5 1 ml ile dolu bir P1000 pipet ucu takın. Paslanmaz çelik harç çekal flushate tampon ve çekal luminal içeriği yıkayın. 1 ml çekal flushate tampon çekal içeriğinin tüm ateş basması için yeterli olacaktır. Çekal flushate içeriğini harç ile temas halinde hemen donar. </li><li> Steril bir havaneli çekal flushate içeriğini Grind. </li><li> Yeri zemin kuru buz / etanol banyosu içinde batık bir 50 ml polipropilen konik tüp içine çekal luminal içeriğini donmuş. </li><li> Her ek fare için 2.7 üzerinden 2.2 adımları tekrarlayın. </li><li> -80 ° C saklayınız </li></ol> 3. Bakteriyel RNA İzolasyon <ol><li> 65 parafilm güvenli bir şekilde kapak ile 50 ml Oak Ridge santrifüj tüpüne bulunan asit kloroform / fenol Sıcak 1 hacmi (yaklaşık 3 ml, iki çekal luminal içeriği son hacmi dayalı) (5:1, v / v) ° C su banyosu. </li><li> 5 dakika için 50 ml polipropilen konik tüp bulunan lizis tamponu 0.5 hacmi (2% SDS, 16 mM EDTA ve 200 mM NaCl) 6 kaynatın. </li><li> Çekal luminal içeriğine kaynar lizis tamponu ekleyin. Homojenize edilir. Periyodik vorteks ile 5 dakika boyunca kaynatın. </li><li> Ekle 100 ° C ila 65 ° C Oak Ridge santrifüj tüpüne asit kloroform / fenol çekal içeriğini / lizis örnek. Parafilm ile Seal kap. </li><li> 65 ° C&#39;de 10 dakika boyunca periyodik olarak vorteks (her 2 dakika). </li><li> 4 ° de 15 dakika boyunca 2.500 g örnek santrifüjleyin. </li><li> 50 ml taze bir Oak Ridge santrifüj dikkatlice sulu faz (beyaz arabirimi herhangi bir kaçınarak) aktarın. Sulu fazın hacmi çekal içeriği, lizis tamponu, asit ve kloroform / fenol toplam hacminin yaklaşık% 50 olacaktır. Eşit miktarda asit kloroform / fenol ekleyin. </li><li> Seal parafilm kapağı ve yüksek hızda karıştırın iyice karıştırın. </li><li> 4 ° de 15 dakika boyunca 2.500 g örnek santrifüjleyin. </li><li> 3,9 için 3,7 Adımlar görünür beyaz, sulu ve organik fazlar arasında arayüz kalmayıncaya kadar tekrarlayın. </li><li> Sulu faz 50 ml taze bir Oak Ridge santrifüj dikkatlice aktarın. Kloroform / izoamil alkol (24:1, v / v) eşit miktarda ekleyin. </li><li> Mühür kapak ve vorteks ile iyice karıştırın. </li><li> 4 ° de 15 dakika boyunca 2.500 g örnek santrifüjleyin. 15 ml polipropilen konik tüp sulu faz (hacmi toplam reaksiyon hacmi yaklaşık% 50 olacak) çıkarın ve eşit hacimde izopropanol ekleyin. </li><li> -20 ° &#39;de en az 2 saat veya gece boyunca inkübe edin. </li><li> 2.500 g örnek için 45 dakika 4 ° santrifüjleyin. Jel gibi pelet bir tüpün dibinde oluşturacaktır. Süpernatantı. </li><li> Pelet 1 ml% 70 etanol buz ile yıkayın. Vorteks. 5 dakika boyunca 4 ° 10,000 g örnek Santrifüj. </li><li> Süpernatantı alın ve 10 dakika boyunca oda sıcaklığında pelet kuruması için tüp ters. </li><li> RNaz içermeyen su 200 ul RNA pelletini tekrar. </li></ol> 4. DNaz Tedavi (Turbo DNA-Free, Uygulamalı Biyosistem) <ol><li> 10X Turbo DNaz tampon ve 2 ul Turbo DNaz 20 ul ekleyin ve hafifçe karıştırın. </li><li> 37 ° de 20 dakika inkübe edin. </li><li> 20 ul yeniden süspanse DNaz inaktivasyon reaktif ekleyin ve iyice karıştırın. </li><li> Ara sıra karıştırarak oda sıcaklığında 5 dakika inkübe edin. </li><li> 1.5 dakika boyunca oda sıcaklığında 10.000 g de santrifüj ve yeni bir mikrofuge&#39;ye boru süpernatantı transfer. </li><li> Bir hacim fenol / kloroform soğuk (4 °) asit ekleyin ve 1 dakika için vorteks ile iyice karıştırın. </li><li> 12.500 g oda sıcaklığında 2 dakika boyunca santrifüjleyin. </li><li> Sulu faz yeni bir tüpe transfer ve 1 hacim kloroform / izoamil alkol (49:1 v / v), girdap ekleyin. </li><li> 12.500 g oda sıcaklığında 2 dakika boyunca santrifüjleyin. </li><li> (Toplam reaksiyon hacminin yaklaşık% 50) sulu faz yeni bir tüpe aktarın. </li><li> 3M sodyum asetat, pH 5.5 0.1 hacim ekleBuz gibi% 100 etanol 2.5 hacimleri. Vorteks. </li><li> En az 2 saat veya -20 ° -80 ° &#39;de 30 dakika inkübe edin. </li><li> 12.000 gr santrifüjleyin az 30 dakika süreyle 4 ° </li><li> Süpernatantı. </li><li> 10.000 g 4 ° &#39;de 5 dakika santrifüj pelet 1 ml% 70 etanol buz ile yıkayın. </li><li> 10 dakika boyunca supernatant ve kuru hava çıkarın. </li><li> RNaz ücretsiz su 100 ul pelletini tekrar. Tekrar süspansiyon 65 ° su banyosu yeri ve pelet çözülür gerekebilir. </li><li> DNaz tedavinin etkinliğini test etmek için, ribozomal protein (veya başka bir referans gen) amplifikasyonu için bir standart RT-PCR reaksiyonu hiçbir RT kontrollerini kullanarak arıtılmış ve arıtılmamış örnekleri olabilir. </li></ol> 5. RNA Temizleme Adım (Qiagen, RNeasy Kiti) <ol><li> Qiagen RNeasy Mini El Kitabı (4. Edition, Nisan 2006) sayfa 56-57 bakın. Gösterildiği gibi uygulayın. </li></ol> 6. Temsilcisi Sonuçlar Bu protokolü kullanarak ele bakteriyel toplam RNA miktarını iki cecums yaklaşık 2-3 mg. Kurtarıldı RNA sonraki qPCR, transkripsiyon profilleme ve RNA Seq deneyler için yeterli nicelik ve nitelik taşımaktadır. RNA saflık rutin 260nm/280nm oranı 7 numunenin ölçümü ile değerlendirilen, henüz bu yöntem hakkında hiçbir bilgi RNA bütünlüğünü sağlar. Agilent Bioanalyzer boyutlandırma, ölçme ve kalite kontrol, DNA, RNA, proteinler ve hücreler için bir Mikroakiskan tabanlı bir platform ve RNA Bütünlük sayısı (RIN) 8 olarak bilinen bir RNA bütünlüğünü metrik kullanır . Bu protokol 260/280 1.7 &#39;den 2.0&#39; ye ve RIN değerleri ≥ 7.0 arasında değişen oranlar üretir RNA ayıklanır. Bu protokol tarafından kurtarıldı bakteriyel RNA Agilent Bioanalyzer analizi bir örnek Şekil 1&#39;de gösterilmiştir. <img src="/files/ftp_upload/3293/3293fig1.jpg" alt="Şekil 1"/> Şekil 1: Agilent Bioanalyzer Elektroferogram ve Pseudomonas aeruginosa toplam RNA örnek jel gibi görüntü izole ve fare çekal içeriği kurtarıldı. RIN 8.0.

<ol>
	<li>Bodey, G. P., Bolivar, R., Fainstein, V., Jadeja, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infections+caused+by+Pseudomonas+aeruginosa.">Infections caused by Pseudomonas aeruginosa.</a> Rev Infect Dis. 5, 279-279 (1983).</li><li>Bertrand, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endemicity,+molecular+diversity+and+colonisation+routes+of+Pseudomonas+aeruginosa+in+intensive+care+units.">Endemicity, molecular diversity and colonisation routes of Pseudomonas aeruginosa in intensive care units.</a> Intensive Care Med. 27, 1263-1268 (2001).</li><li>Koh, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+in+vivo+transcription+profiling+for+identifying+Pseudomonas+aeruginosa+genes+needed+for+gastrointestinal+colonization+and+dissemination.">Utility of in vivo transcription profiling for identifying Pseudomonas aeruginosa genes needed for gastrointestinal colonization and dissemination.</a> PLoS One. 5, e15131-e15131 (2010).</li><li>Koh, A. Y., Priebe, G. P., Pier, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virulence+of+Pseudomonas+aeruginosa+in+a+murine+model+of+gastrointestinal+colonization+and+dissemination+in+neutropenia.">Virulence of Pseudomonas aeruginosa in a murine model of gastrointestinal colonization and dissemination in neutropenia.</a> Infect Immun. 73, 2262-2272 (2005).</li><li>Alexander, R. J., Raicht, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+total+RNA+from+human+stool+samples.">Purification of total RNA from human stool samples.</a> Dig Dis Sci. 43, 2652-2658 (1998).</li><li>Fitzsimons, N. A., Akkermans, A. D., de Vos, W. M., Vaughan, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+gene+expression+detected+in+human+faeces+by+reverse+transcription-PCR.">Bacterial gene expression detected in human faeces by reverse transcription-PCR.</a> J Microbiol Methods. 55, 133-140 (2003).</li><li>Manchester, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+UV+methods+for+measurement+of+protein+and+nucleic+acid+concentrations.">Use of UV methods for measurement of protein and nucleic acid concentrations.</a> Biotechniques. 20, 968-970 (1996).</li><li>Schroeder, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RIN:+an+RNA+integrity+number+for+assigning+integrity+values+to+RNA+measurements.">The RIN: an RNA integrity number for assigning integrity values to RNA measurements.</a> BMC Mol Biol. 7, 3-3 (2006).</li><li>Turnbaugh, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+core+gut+microbiome+in+obese+and+lean+twins.">A core gut microbiome in obese and lean twins.</a> Nature. 457, 480-484 (2009).</li></ol>

Çıkar çatışması ilan etti.

RNA ekstraksiyon yöntemi, burada açıklanan kurtarma için fare GI yolu hasat yüksek kaliteli Pseudomonas aeruginosa toplam RNA yeterli miktarda sağlar . Bu yöntem, P. ile sınırlı değildir aeruginosa ve diğer bakterilerin potansiyel olarak uygulanabilir. Yeterli mikrobik organizmaların bağırsak kurtarma organizmadan organizmaya önemli ölçüde değişecektir. Bizim fare modeli, P. aeruginosa genellikle 5 x 10 7 5 x 10 8 kob / g feçes 4 arasındaki seviyelerde fare gastrointestinal sistem kolonize. Kurtarılan çekal içeriği kabaca 0.5 gram, P. tahmini sayısı olduğundan iki cecums kurtarıldı aeruginosa 5 x 10 7 5 x 10 8 kob arasında. Diğer mikroorganizmaların kullanılması halinde, gastrointestinal kolonizasyon seviyesini doğrulamak ve daha sonra hedeflenen sayıda kob kurtarmak için gerekli cecums sayısını hesaplamak için ihtiyatlı bir davranış olacaktır. Bu özel fare modeli kullanırken, PA ile enfekte olmayan antibiyotik ile tedavi edilen farelerde çekal içeriğini izole edilen RNA ölçülebilir miktarda olduğunu da not etmek önemlidir. Pseudomonas aeruginosa gastrointestinal kolonizasyon ve yaygınlaştırma girişimleri P. patogenezinde taklit etmek için fare modeli kanser ve kök hücre nakli yapılan hastaları bakteriyemi aeruginosa . Bu hasta popülasyonunda, ortakçı florasının genellikle antibiyotik veya kemoterapötik tedavi (GI ortakçı floranın antibiyotik tükenmesi gibi) patojen mikropların (örneğin P. aeruginosa ile mono-dernek) aşırı üremesine neden tükenmiş ve bağışıklık baskılanması sonra sonraki yayılması. Bu fare modelinin avantajları ve sınırlamaları zaten daha önce 4 olarak ele alınmıştır . Bu çalışmanın amacı, GI yolu mikrobik total RNA izole etmek için bir metodoloji sunmaktır. Bu protokol, gastrointestinal sistem (yani ortakçı flora etkileşimleri, inflamatuvar barsak hastalığı gibi bakteriyel etkileri) mikrobiyal patogenez diğer yönlerini incelemek diğer fare modelleri kolayca adapte edilebilir. Bu yöntemin bir avantajı, donma / çözülme, mekanik parçalama (harç / havaneli, homojenizasyon ile ezerek), kaynama, kimyasal ve lizis (örneğin SDS) de dahil olmak üzere birden fazla lizis adımları birleşme olduğunu. Lizis adımları çokluğuna rağmen, bazı mikroorganizmaların (özellikle Gram-pozitif bakteri ve mantar / maya) ilave mekanik bozulması gerekebilir. Sıcak lizis / asit, fenol-kloroform inkübasyon (Adım 3.5), boncuk ek (bakteri ve maya / veya 0.5-0.7 mm 0,1 mm) ve bir sonraki boncuk atan adım sonra, bu organizmalar 9 lyse için yeterli olmalıdır 10 Fare çekum, tekrarlayan soğuk acid-phenol/chloroform ekstraksiyon (Adım 3,7-3,9) kurtarıldı malzemelerin karmaşık doğası gereği kesinlikle daha aşağı reaksiyonlar için kabul edilebilir RNA kalitesini ve bütünlüğünü sağlamak için gerekli. 3-5 soğuk acid-phenol/chloroform ekstraksiyon arasında elimine edilir, sulu ve organik fazlar arasında beyaz bir arayüz önce gerekli olabilir. Son olarak, DNaz tedavi (Adım 4) ve RNeasy Temizleme Protokolü (Adım 5) kombinasyonu kirlenmesine neden olan DNA ve mRNA küçük olmayan (5s ve tRNA) kaldırmak için gereklidir. Daha önce de belirtildiği gibi, bu protokol kullanan tarafından kurtarıldı RNA sonraki transkripsiyon profil 3, qPCR (yayımlanmamış), ve RNA Seq (yayımlanmamış) deneyler için yeterli miktar ve kalitede.

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th></tr><tr><td> Havanda </td><td> Balıkçı </td><td> 12-947-1 </td></tr><tr><td> Homojenizatör </td><td> Omni </td><td> TM-125 </td></tr><tr><td> Oak Ridge santrifüj tüplerine (50 ml) </td><td> Nalgene </td><td> 3119-0050 </td></tr><tr><td> Asit Fenol / Kloroform </td><td> Ambion </td><td> AM9720 </td></tr><tr><td> Kloroform </td><td> Sigma-Aldrich </td><td> C2432 </td></tr><tr><td> Izoamil Alkol </td><td> Sigma-Aldrich </td><td> W205702 </td></tr><tr><td> Isopropanol </td><td> Sigma-Aldrich </td><td> 190764 </td></tr><tr><td> Dietil pyrocarbonate (DEPC) </td><td> Sigma-Aldrich </td><td> 40718 </td></tr><tr><td> DNaz </td><td> Ambion </td><td> AM2238 </td></tr><tr><td> RNeasy Seti </td><td> Qiagen </td><td> 74104 </td></tr><tr><td> 3M Sodyum Asetat </td><td> Ambion </td><td> AM9740 </td></tr><tr><td> % 100 Etanol </td><td> Sigma-Aldrich </td><td> E7023 </td></tr></tbody></table>

rna isolation of pseudomonas aeruginosa colonizing the murine gastrointestinal tract

Lösemili hastalarda, ağır yanık yaraları, veya organ nakli 1 gibi bağışıklık sistemleri ile ev sahipliği yapan önemli bir morbidite ve mortalite Pseudomonas aeruginosa (PA) enfeksiyonları sonucu. PA kan dolaşımı enfeksiyonu geliştirme için yüksek riskli hastalarda, gastrointestinal (Gİ) sistem 2 kolonizasyon için ana rezervuar, ancak mekanizmaları olan asemptomatik kolonize mikrop invaziv, ve genellikle ölümcül PA geçişler, patojen belirsizdir. Daha önce, konağın bağışıklık durumu değişiklikler sırasında bakteriyel gen ekspresyonu değişiklikleri belirlemek amacıyla fareler 3 kolonize cecums yaşayan bakteri hücrelerinin PA mRNA kurtarma in vivo transkripsiyon profili deneyleri yapıldı. Herhangi bir transkripsiyon profil deneyde olduğu gibi, hız sınırlayıcı adım yeterli miktarda yüksek kaliteli mRNA izolasyonu. Enzimler, moloz, gıda artıkları ve GI kanalda partikül madde bolluğu göz önüne alındığında, RNA izolasyonu meydan yıldırıcı. Burada, biz, güvenilir ve tekrarlanabilir fare GI sistem kurtarıldı bakteriyel RNA izolasyonu için bir yöntem mevcut. Bu yöntem, PA gastrointestinal kolonizasyon ve nötropeni indüklenen yayılması 4 köklü bir fare modeli kullanmaktadır. PA ile bir kez gastrointestinal kolonizasyon doğruladı, fareler, ötenazi ve çekal içeriğini kurtarıldı ve flash dondurulur. RNA sonra kaynar bozulması, mekanik, fenol / kloroform ekstraksiyon, DNaz tedavi ve afinite kromatografisi bir kombinasyonu kullanılarak elde edilir. Miktar ve saflık spektrofotometri (Nanodrop Teknolojileri) ve bioanalyzer (Agilent Technologies) (Şekil 1) tarafından teyit edilir. GI mikrobiyal RNA izolasyonu Bu yöntem yanı sıra diğer bakteri ve mantarlar kolayca adapte edilebilir.

Watch this Scientific Journal Video about RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract at JoVE.com

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

RNA izolasyonu için güvenilir bir yöntem<em&gt; Pseudomonas aeruginosa</em&gt; Fare cecums kurtarıldı açıklanmıştır. Kurtarıldı RNA sonraki qPCR, transkripsiyon profilleme ve RNA Seq deneyler için yeterli nicelik ve nitelik taşımaktadır. Bu teknik diğer bağırsak mikropların RNA izolasyonu için adapte edilebilir.

RNA İzolasyonu Pseudomonas aeruginosa Murin Gastrointestinal Yolu koloniler

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

rna-isolation-pseudomonas-aeruginosa-colonizing-murine

Research

JoVE Journal

Immunology and Infection

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

19.1K Görüntüleme.  University of Texas Southwestern Medical Center. RNA izolasyonu için güvenilir bir yöntem Pseudomonas aeruginosa Fare cecums kurtarıldı açıklanmıştır. Kurtarıldı RNA sonraki qPCR, transkripsiyon profilleme ve RNA Seq deneyler için yeterli nicelik ve nitelik taşımaktadır. Bu teknik diğer bağırsak mikropların RNA izolasyonu için adapte edilebilir.

Video: RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

Co-kültür Modelleri Pseudomonas aeruginosa</emCanlı İnsan Havayolu Hücreler Yetiştirilen> Biyofilmler

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

İmmünoloji ve Enfeksiyon

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

Pseudomonas aeruginosa ve Saccharomyces cerevisiae Biyofilm Akış Hücreler

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Fare Genital Sistemlerinin Mukozanin gelen Lenfosit İzolasyonu

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

Uzun Vadeli Kronik<em&gt; Pseudomonas aeruginosa</emFarelerde&gt; Havayolu Enfeksiyon

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

Pseudomonas aeruginosa Kaynaklı Akciğer Hasarı Modeli

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Here we describe a novel diabetic murine model utilizing hairless mice for real-time, non-invasive, monitoring of biofilm wound infections of bioluminescent Pseudomonas aeruginosa. This method can be adapted to evaluate infection of other bacterial species and genetically modified microorganisms, including multi-species biofilms, and test the efficacy of antibiofilm strategies.

Işık tarafa: In Vivo izleme bir diyabetik tüysüz fare modelinde kronik yaralarda Pseudomonas aeruginosa biyofilm enfeksiyonların

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

Sipariş edilmiş, Pseudomonas aeruginosa baskı PA14 Transposon ekleme mutantlar Nonredundant Kütüphane çoğaltılması

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

Yöntemleri için algılama sitotoksik Amyloids aşağıdaki enfeksiyon pulmoner endotel hücre Pseudomonas aeruginosa tarafından

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

Situ Hibridizasyonfloresan Kullanarak Murine Gastrointestinal Sistemde Candida albicans Görselleştirilmesi

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype called mucoidy. Alginate is linked to chronic lung infections resulting in poor prognosis in patients with cystic fibrosis (CF). Understanding the pathways that regulate the production of alginate can aid in the development of novel therapeutic strategies targeting the alginate formation. Another disease-related phenotype is the small colony variant (SCV). SCV is due to the slow growth of bacteria and often associated with increased resistance to antimicrobials. In this paper, we first show a method of culturing a genetically defined form of P. aeruginosa SCV due to pyrimidine biosynthesis mutations. Supplementation of nitrogenous bases, uracil or cytosine, returns the normal growth to these mutants, demonstrating the presence of a salvage pathway that scavenges free bases from the environment. Next, we discuss two methods for the measurement of bacterial alginate. The first method relies on the hydrolysis of the polysaccharide to its uronic acid monomer followed by derivatization with a chromogenic reagent, carbazole, while the second method uses an ELISA based on a commercially available, alginate-specific mAb. Both methods require a standard curve for quantitation. We also show that the immunological method is specific for alginate quantification and may be used for the measurement of alginate in the clinical specimens.

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Here, we describe a growth condition to culture the small colony variant of Pseudomonas aeruginosa. We also describe two separate methods for the detection and quantitation of the exopolysaccharide alginate produced by P. aeruginosa using a traditional uronic acid carbazole assay and an alginate-specific monoclonal antibody (mAb) based ELISA.

Pseudomonas aeruginosa Küçük Koloni Varyant Kültürü ve Aljinat Quantitation

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype ...

Methods Article

RNA İzolasyonu Pseudomonas aeruginosa Murin Gastrointestinal Yolu koloniler